About

Susan Hafenstein, PhD, is the Director of cryoEM at the Hormel Institute and a Professor. Her research focuses on understanding the structure and functions of viruses, specifically host-virus interactions that facilitate viral entry into cells. She is an expert in cryo-electron microscopy (cryoEM), utilizing local reconstruction approaches to solve atomic resolution maps of viruses such as HPV, murine papillomavirus, polyomavirus, and Zika virus. Dr. Hafenstein joined the Hormel Institute in 2023 and contributes to advancing structural virology through her specialized expertise in cryoEM techniques.

Research topics

• Computational biology
• Biology
• Chemistry
• Computer Science
• Artificial Intelligence
• Virology
• Genetics
• Cell biology
• Medicine
• Biochemistry
• Physics

Selected publications

• Transferrin receptor 1 binds human parvovirus B19 VP1u to facilitate entry

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-03-13

  articleOpen accessCorresponding

  Summary Human parvovirus B19 (B19V) exhibits a strict tropism for erythroid progenitor cells, which is governed by the VP1 unique region (VP1u). This region mediates cell-specific uptake by interacting with an unknown cellular receptor, termed VP1uR. Proximity labeling in permissive erythroid cells identified transferrin receptor 1 (TfR1/CD71) as the predominant membrane protein associated with VP1u. VP1u constructs colocalized with TfR1 at the cell surface of erythroid cells. Incubation with anti-TfR1 antibody OKT9 abolished binding and uptake of recombinant VP1u. While OKT9 efficiently inhibited B19V uptake and infection, it did not block virus binding to host cells. Direct binding assays confirmed interaction of VP1u to human TfR1. Using cryoEM we solved the 2.4 Å structure of the TfR1-VP1u complex, mapping the binding site and identifying the specific interactions. These findings establish TfR1 as the previously unknown receptor, VP1uR, required for B19V uptake.

  PublisherOA PDFDOI

• Structural Basis for C ⁸ methylation of 23S ribosomal RNA by Cfr

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-03-02

  articleOpen access

  Abstract Cfr methylates C 8 of adenosine 2503 (A2503) in 23 S ribosomal RNA (rRNA) and will also methylate C 2 of A2503 after methylating C 8 . C 8 methylation confers resistance to more than five classes of clinically used antibiotics, highlighting it as a worrisome mechanism of antibiotic resistance. Here, we report the structure of Cfr, determined by cryogenic electron microscopy (Cryo-EM). Despite its small size (∼36 kDa), we exploit a transient protein–RNA crosslink that forms during catalysis, which requires Cys105 to resolve. Using a Cfr Cys105Ala variant and an 87-nucleotide strand of rRNA, we isolate the crosslinked species and determine its structure to 3.0 Å resolution. Notably, the 87-mer rRNA adopts an L-shaped conformation characteristic of tRNAs, rather than the conformation it assumes in the ribosome. One Sentence Summary Cryo-EM structure of Cfr, a radical S-adenosylmethionine methylase that confers antibiotic resistance

  PublisherDOI

• Neutralizing human monoclonal antibodies to poliovirus map to the receptor binding site

  Nature Communications · 2026-01-02

  articleOpen accessSenior author

  Poliovirus remains a serious threat to human health. Complete eradication of wild-type poliovirus has not yet succeeded, making the development of successful antivirals critical. Microneutralization assays against all three poliovirus serotypes identified a panel of human monoclonal IgGs, which are either serotype-specific or cross-neutralizing. Here, through cryoEM single particle analysis, we solved high resolution structures of four distinct poliovirus-FAb complexes. These antibodies bind to capsids at the circular depression (canyon) surrounding the icosahedral five-fold symmetry axis, which is also the binding site of the poliovirus receptor (PVR). Analysis of these structures confirms overlap of FAb contacts on the viral capsid with those of PVR. For three of the FAbs, the capsid residues are identified that dictate serotype-specific recognition. Contacts for the cross-neutralizing mAb 10D2 are located deep in the capsid canyon. These structural analyses indicate that antibody competition with the receptor likely leads to neutralization of virus particles and inhibition of poliovirus entry into host cells. Thus, the human IgGs studied here may facilitate development of therapeutics for the ongoing efforts in global eradication of poliovirus.

  PublisherDOI

• Structures and functions of the limited natural polyclonal antibody response to parvovirus infection

  Proceedings of the National Academy of Sciences · 2025-02-14 · 4 citations

  articleOpen accessCorresponding

  Host antibody responses are key components in the protection of animals against pathogens, yet the defining properties of viral antigens and induction of B cell responses that result in varied protection are still poorly understood. Parvoviruses are simple molecular structures that display 60 repeated motifs on their capsid surface, and rapidly induce strong antibody responses that protect animals from infection. We recently showed that following canine parvovirus infection of its natural host, the polyclonal response in the sera contained only two or three dominant antibodies that bound two epitopes on the capsid. Here, we characterize key antibodies present in that immune response, identifying their sequences, defining their binding properties on the capsid by cryoelectron microscopic (cryoEM) analysis, and testing their effects on viral infectivity. Two antibodies sharing the same heavy chain bound to the side of the capsid threefold spike (B-site), while another distinct antibody bound close to the threefold axis (A-site). The epitopes of these antibodies overlapped the binding site of the host receptor, the transferrin receptor type-1, but to varying degrees. The antibodies varied widely in their neutralization efficiencies as either immunoglobulins (IgGs) or monomeric antigen-binding fragments (Fabs), which was consistent with their ability to compete for the receptor. The monoclonal antibodies characterized here matched the structures from the cryoEM analysis of polyclonal sera, including those present in a different dog than the monoclonal source. This shows that after infection, a focused response to the viral antigen is produced that protects against infection.

  PublisherDOI

• Distinct evolutionary patterns of endemic and emerging parvoviruses and the origin of a new pandemic virus

  Proceedings of the National Academy of Sciences · 2025-07-18

  preprintOpen access

  ABSTRACT Emergence of epidemic viruses in new hosts threatens both human and animal populations, and often involves virus evolution to overcome barriers that normally prevent efficient infection and spread in that host. After transfer the separated viruses will evolve in parallel as they spread within the original and new hosts. Here we examine the details of a virus involved in such a host-jumping event, where we define the natural evolution of feline panleukopenia virus (FPV) over 60 years, clarify the origins of the new pandemic canine parvovirus (CPV) that arose in the 1970s, and compare the separate evolution of those viruses over 47 years in cats or dogs. Several live-attenuated FPV vaccine viruses originated from early 1960s isolates or were a recombinant of an early virus, and many sequences in databases proved to be vaccine-derived. The sequences of wild viruses showed that FPV-like strains evolved at ∼25% the rate seen for CPV in dogs, and the higher rate of CPV evolution was consistent since 1979 when a genetic variant became widespread. The common ancestor of the CPV lineage was related to FPVs from Europe, and contained several unique host-adaptive capsid changes associated with canine transferrin receptor type-1 binding. Although the FPV vaccine strains are around 60 years old, little selection for antigenic variation was observed. The distinct evolutionary patterns of these closely related viruses circulating for decades in different hosts emphasizes the complex evolution associated with viral epidemic emergence and spread in endemic and new hosts. SIGNIFICANCE STATEMENT Comparing the evolution of a virus in its reservoir host with that seen in a new host will reveal the special circumstances that allow epidemic emergence. A feline parvovirus (FPV) jumping to dogs in the mid-1970s formed canine parvovirus (CPV), which has circulated world-wide until today. The evolutionary rate of FPV in its original hosts was much lower than that of CPV in dogs, and the mutational patterns seen in the different hosts were also distinct. Early CPV isolates differed from the ancestral FPV clade in several key host range mutations. These results highlight the complex biology associated with epidemic emergence, including host-specific rates of lineage evolution and complex origins of host-adaptive mutations. (113/120)

  PublisherDOI

• Abstract 2655 Functional and Structural Characterization of F420H2 and Ferredoxin-Dependent Heterodisulfide Reductase Subunit A: A New Class of Electron-Bifurcating Enzymes

  Journal of Biological Chemistry · 2025-05-01

  articleOpen access

  Electron bifurcation is a vital energy conservation mechanism that allows organisms to drive thermodynamically unfavorable reactions without relying solely on ATP hydrolysis or ion gradients. This process involves splitting a pair of electrons from a single donor, directing one to a high-potential acceptor and the other to a low-potential acceptor. While flavin-based electron bifurcation systems using NAD(H), NADP(H), or H2 as low-potential electron sources are well-characterized, the role of F420H2 as an electron donor in bifurcation remains poorly understood.

  PublisherOA PDFDOI

• Atomic-resolution structure of a chimeric Powassan tick-borne flavivirus

  Science Advances · 2025-07-09 · 2 citations

  articleOpen accessSenior authorCorresponding

  Powassan virus (POWV) is an emerging tick-borne flavivirus for which no vaccine or antiviral treatment exists. The incidence of human infections of POWV in North America has been increasing because of the expanding distribution of the tick vector Ixodes scapularis that transmits POWV to humans. To mitigate the dangers of handling a risk group 3 human pathogen, a chimeric virus was constructed from the genetic backbone of a yellow fever virus vaccine strain 17D (YFV-17D) and the external structural proteins of POWV lineage II. The chimera had a comparable phenotype as POWV. The atomic resolution of yellow fever virus-Powassan virus chimera (yPOWV) structure was built without ambiguity, revealing surface glycans and lipid pocket factors. The similarity to other flavivirus structures and the phenotype similar to YFV-17D suggest that there could be future potential as a vaccine candidate.

  PublisherDOI

• The CD4 T cell-independent IgG response during persistent virus infection favors emergence of neutralization-escape variants

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-12-25 · 4 citations

  preprintOpen access

  How changes in the quality of anti-viral antibody (Ab) responses due to pre-existing or acquired CD4 T cell insufficiency affect virus evolution during persistent infection are unknown. Using mouse polyomavirus (MuPyV), we found that CD4 T cell depletion before infection results in short-lived plasma cells secreting low-avidity antiviral IgG with limited BCR diversity and weak virus-neutralizing ability. CD4 T cell deficiency during persistent infection incurs a shift from a T-dependent (TD) to T-independent (TI) Ab response, resembling the pre-existing TI Ab response. CD4 T cell loss before infection or during persistent infection is conducive for emergence of Ab-escape variants. Cryo-EM reconstruction of complexes of MuPyV virions with polyclonal IgG directly from infected mice with pre-existing or acquired CD4 T cell deficiency enabled visualization of shortfalls in TI IgG binding. By debilitating the antiviral IgG response, CD4 T cell deficiency sets the stage for outgrowth of variant viruses resistant to neutralization.

  PublisherDOI

• Infectious parvovirus B19 circulates in the blood coated with active host protease inhibitors

  Nature Communications · 2024-11-05 · 2 citations

  articleOpen accessCorresponding

  The lack of a permissive cell culture system has limited high-resolution structures of parvovirus B19 (B19V) to virus-like particles (VLPs). In this study, we present the atomic resolution structure (2.2 Å) of authentic B19V purified from a patient blood sample. There are significant differences compared to non-infectious VLPs. Most strikingly, two host protease inhibitors (PIs), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) and serpinA3, were identified in complex with the capsids in all patient samples tested. The ITIH4 binds specifically to the icosahedral fivefold axis and serpinA3 occupies the twofold axis. The protein-coated virions remain infectious, and the capsid-associated PIs retain activity; however, upon virion interaction with target cells, the PIs dissociate from the capsid prior to viral entry. Our finding of an infectious virion shielded by bound host serum proteins suggests an evolutionarily favored phenomenon to evade immune surveillance and escape host protease activity. This study presents the first high-resolution structure of infectious parvovirus B19, purified from a patient. Two acute phase reactants, serpinA3, and ITIH4, bind to the capsid, suggesting B19V utilizes host proteins during viremia for survival and function.

  PublisherOA PDFDOI

• Intranasal SARS-CoV-2 RBD decorated nanoparticle vaccine enhances viral clearance in the Syrian hamster model

  Microbiology Spectrum · 2024-02-09 · 10 citations

  articleOpen access

  Multiple vaccines have been developed and licensed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). While these vaccines reduce disease severity, they do not prevent infection. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor-binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4 A using cryogenicelectron microscopy. Using this RBD-grafted SpyCage scaffold (RBD + SpyCage), we performed two intranasal vaccination studies in the "gold-standard" pre-clinical Syrian hamster model. The initial study focused on assessing the immunogenicity of RBD + SpyCage combined with the LTA1 intranasal adjuvant. These studies showed RBD + SpyCage vaccination induced an antibody response that promoted viral clearance but did not prevent infection. Inclusion of the LTA1 adjuvant enhanced the magnitude of the antibody response but did not enhance protection. Thus, in an expanded study, in the absence of an intranasal adjuvant, we evaluated if covalent bonding of RBD to the scaffold was required to induce an antibody response. Covalent grafting of RBD was required for the vaccine to be immunogenic, and animals vaccinated with RBD + SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens.IMPORTANCEDespite the availability of efficacious COVID vaccines that reduce disease severity, SARS-CoV-2 continues to spread. To limit SARS-CoV-2 transmission, the next generation of vaccines must induce immunity in the mucosa of the upper respiratory tract. Therefore, we performed proof-of-principle, intranasal vaccination studies with a recombinant protein nanoparticle scaffold, SpyCage, decorated with the RBD of the S protein (SpyCage + RBD). We show that SpyCage + RBD was immunogenic and enhanced SARS-CoV-2 clearance from the nose and lungs of Syrian hamsters. Moreover, covalent grafting of the RBD to the scaffold was required to induce an immune response when given via the intranasal route. These proof-of-concept findings indicate that with further enhancements to immunogenicity (e.g., adjuvant incorporation and antigen optimization), the SpyCage scaffold has potential as a versatile, intranasal vaccine platform for respiratory pathogens.

  PublisherDOI

Recent grants

• NIH Grant F32AI060155

  NIH · $142k · 2007

• Mechanisms of Enterovirus Entry

  NIH · $3.9M · 2014–2026

• Structural Studies of Virus and Receptor Interaction

  NIH · $258k · 2009–2011

Frequent coauthors

• Carol M. Bator

  Pennsylvania State University

  36 shared

• Hyunwook Lee

  Hormel (United States)

  34 shared

• Colin R. Parrish

  Cornell University

  33 shared

• Maria Gullberg

  28 shared

• Nina Jönsson

  Norwegian Institute for Nature Research

  28 shared

• Anders Lindberg

  Royal North Shore Hospital

  28 shared

• Daniel J. Goetschius

  28 shared

• Conny Tolf

  27 shared

Education

• PhD

  University of Arizona

  2003

Awards & honors

• Dr. James E. Rubin Medical Memorial Award
• Graduating Medical Student Research Award
• Veneziale-Steer Award
• Dr. Marvin and Hadassah Bacaner Research Awards
• Schmidt Steer Award